33.2.27
Maxi-
AOAC Official Method 989.04
Fat in Raw Milk
Babcock Method
Final Action 1920
Revised First Action 1989
Revised First Action 1997
Final Action 2000
(Applicable to raw, whole milk.)
Results of the interlaboratory study supporting the acceptance of the
method (for sections A–D):
sr = 0.037; sR = 0.047; RSDr = 0.901%; RSDR = 1.147%; r = 0.105;
R = 0.133
A. Principle
Known weight of milk is placed into Babcock bottle and H2SO4 is
added. Generated heat releases fat, which is then isolated by centri-
fuging and addition of H2O. Fat is quantitated in graduated portion
of Babcock bottle. Result is expressed as % fat by weight.
B. Reagents
(a) Sulfuric acid (H2SO4).—Specific gravity 1.82–1.83 at 20°C.
(b) Mercury (Hg).—Purity ≥99.99%. Used for testing accuracy
of Babcock bottles.
C. Apparatus
(a) Standard Babcock milk-test bottle.—8%, 18 g milk-test bot-
tle, total height 160–170 mm (6.3–6.7 in.). Bottom of bottle is flat,
and axis of neck is vertical when bottle stands on level surface.
(1) Bulb.—Capacity of bulb to junction with neck is ≥45 mL.
Bulb may be either cylindrical or conical. If cylindrical, od of base
must be 34–36 mm; if conical, od of base must be 31–33 mm, and
maximum diameter, 35–37 mm.
(2) Neck.—Cylindrical and of uniform diameter from ≥5 mm be-
low lowest graduation mark to ≥5 mm above highest. Top of neck is
flared to diameter of ≥10 mm. Graduated portion of neck is ≥75 mm
long and is graduated in whole %, 0.5%, and 0.1% from 0.0 to 8.0%.
Graduations may be etched, with black or dark pigment annealed to
graduation, or may be unetched black or dark lines permanently an-
nealed to glass. Graduation line widths ≤0.2 mm. Tenth % gradua-
tions are ≥3 mm long; 0.5% graduations are ≥4 mm long and project
1 mm to left; and whole % graduations extend at least half way
around neck to right and project ≥2 mm to left of 0.1% graduations.
Each whole % is numbered, with numeral placed to left of scale.
Vertical line must be etched and annealed with black or dark pig-
ment or must be etched into dark stripe permanently annealed to
glass. Etched vertical line is located 1 mm to right of 0.1% gradua-
tion marks and extends ≥1 mm above 8% line and ≥1 mm below 0%
line. If vertical line is etched into dark vertical stripe, then dark verti-
cal stripe must extend ≥1 mm above the 8% line and ≥1 mm below
0% line. Dark vertical stripe should be ≤2.0 mm wide and etched line
must be ≤0.2 mm wide.
Zero line (≤0.2 mm wide) must be etched and annealed with black
or dark pigment, or not darkened but etched into dark horizontal
stripe (≤2.0 mm wide) permanently annealed to glass. Zero and ver-
tical etched lines, with or without annealed pigment, should be of
sufficient depth to hold point of dividers.
Capacity of neck for each whole % on scale is 0.200 mL.
mum error of total graduation or any part thereof must not exceed
0.008 mL.
(3) Testing.—Accuracy of each bottle shall be determined before
use (usually by manufacturer or certified laboratory). Bottle calibra-
tion accuracy is determined by placing bottle upside down on Bab-
cock bottle calibration apparatus (Nafis tester or modified Nafis
tester) that is capable of delivering known volumes of Hg into Bab-
cock bottle neck. Bottle calibration apparatus delivery is calibrated
and volume of Hg contained between 8 and 4% (0.800 mL), 4 and
0% (0.800 mL), and 8 and 0% (1.600 mL) marks is determined. Ac-
curacy of bottle neck calibration may be determined before or after
attachment to bulb. Accuracy should be at least ±0.04% on scale of
18 g milk-test bottle. [Reference: J. AOAC Int. 78, 463(1995).]
(b) Pipet.—Standard milk pipet conforms to following
specifications:
Total length . . . . . . . . . . . . . . . . . . . . . . . ≤ 350 mm
OD of suction tube . . . . . . . . . . . . . . . . . . . . . 6–8 mm
Length of suction tube . . . . . . . . . . . . . . . . . . . 130 mm
OD of delivery tube [must fit into bottle, (a)] . . . . . 4.5–5.0 mm
Length of delivery tube
(slightly longer than bottle neck, must not touch
milk test portion when delivered) . . . . . . . . . 105–125 mm
Distance of graduation mark above bulb . . . . . . . . 15–45 mm
Nozzle parallel with axis of pipet, but slightly
constricted so as to discharge in 5–8 s when filled
with H2O
Graduation, marked to contain 17.6 mL H2O at 20°C
when bottom of meniscus coincides with mark on
suction tube
Maximum error in graduation . . . . . . . . . . . . . . ≤0.05 mL
To test pipet, place tip of pipet against firm rubber surface, clamp
pipet in vertical position, and use buret (Class A—graduations
≤0.05 mL) to fill pipet to graduation mark with H2O (at 20°C).
(c) Acid measure.—Device used to measure H2SO4 should de-
liver in range from 10 to 20 mL. Use device that can be set to consis-
tently deliver appropriate amount of acid to obtain desired milk acid
reaction temperature in D.
(d) Centrifuge or “tester.”—Standard centrifuge, however
driven, must be constructed throughout and so mounted as to be ca-
pable, when filled to capacity, of rotating at necessary speed with
minimum vibration and without liability of causing injury or acci-
dent. Centrifuge must be heated, electric or otherwise, to 46–51°C
during centrifuging. It must be provided with speed indicator, per-
manently attached, if possible. Proper rate of rotation may be deter-
mined by reference to Table 989.04. Rotation speed with full
centrifuge should be checked periodically with tachometer. “Diam-
eter of wheel” means distance between inside bottoms of opposite
cups measured through center of rotation of centrifuge wheel while
cups are horizontally extended. Relative centrifugal force is approx-
imately 164 × g.
(e) Dividers or calipers.—For measuring fat column.
(f) Water bath for test bottles.—Provided with thermometer and
device to maintain temperature of fat column at 48 ± 1°C.
(g) Water bath for tempering milk samples prior to weigh-
ing.—Provided with thermometer and device to maintain tempera-
ture of milk at 38 ± 1°C.
© 2002 AOAC INTERNATIONAL
 (h) Supply of hot water added after 1st and 2nd
centrifugations.—Provided with thermometer, device to maintain
temperature of distilled H2O at 50–52°C, and device to deliver H2O
into Babcock bottles.
(i) Bottle shaker.—Variable speed and matched to maximum ca-
pacity of centrifuge.
(j) Digital thermometer for measurement of milk-acid reaction
temperature.—Capable of reading temperature to the nearest degree
in range of 100–120°C. Use acid-resistant probe, with small diame-
ter (≤0.5 mm) to ensure rapid response time. Length of temperature
probe should be such that its tip is ca 1 cm above bottom of bottle,
when fully inserted.
(k) Reading light with magnifying glass.—As background when
measuring fat columns. Light should be diffused (soft white color)
and provide illumination from angles above and below level of fat
column. Magnification device must be used to aid reading.
(l) Tachometer.—For determining rotation speed of centrifuge
(capable of measuring between 600–1000 rpm).
D. Determination
(a) Test portion preparation and temperature adjust-
ment.—Place sample in H2O bath at 38 ± 1°C. Level of H2O should
be at or above milk level. Mix test portion 10× by inversion. If fat
line remains on inside surface of container, run hot tap H2O (ca
50–60°C) over outside surface for 15–20 s. Mix test portion thor-
oughly by inversion and pipet immediately. Do not allow test por-
tions to remain in H2O bath more than 15 min after reaching 38°C.
With pipet, C(b), transfer 17.6 ± 0.05 mL prepared test portion at
38°C to milk-test bottle. Also, transfer 17.6 ± 0.05 mL milk at 38°C
to another milk-test bottle for measurement of milk-acid reaction
temperature. Blow out milk in pipet tip ca 30 s after free outflow
ceases. Adjust temperature of milk in bottle to 20–22°C. Adjust tem-
perature of H2SO4 to 20–22°C.
(b) Measurement of milk-acid reaction temperature and deter-
mination of amount of H2SO4 to use.—Before testing group of test
samples, determine correct amount of acid to be used in analysis by
measuring milk-acid reaction temperature. Add 17.5 mL H2SO4 to
bottle from (a) containing tempered milk test portion prepared for
measurement of milk-acid reaction temperature. Add acid in one de-
livery that washes all traces of milk into bulb and cleanly layers acid
under milk. Fully insert digital thermometer probe down bottle neck
and immediately shake by hand rotation until all traces of curd disap-
pear. Peak reaction temperature should be 108 ± 2°C. Adjust amount
of H2SO4 added, until reaction temperature is within this range and
fat column is translucent golden-yellow to amber. Amount of acid
required may vary between technicians and between batches of acid.
(c) Testing milk test portions.—To test bottle from (a) containing
tempered milk, add appropriate amount of H2SO4 [as determined in
(b)] by delivering acid in one delivery that washes all traces of milk
into bulb and cleanly layers acid under milk. Immediately shake bot-
tle by hand rotation as in (b), until all traces of curd disappear. Place
bottle in Babcock bottle shaker set at medium speed. Continue to add
acid to all samples, and then shake full set 1 additional min. Temper-
ature of milk plus acid in first bottle should not be less than 50.5°C at
time bottles are transferred to centrifuge.
Place bottles in heated centrifuge, counter-balance, and centrifuge
5 min after proper speed is reached. Add distilled H2O at 50–52°C
until bulb of bottle is filled. Centrifuge 2 min. Add distilled H2O at
50–52°C until top of fat column approaches 7% mark on bottle neck.
Centrifuge 1 min longer at ca 51°C. Transfer bottle to H2O bath
Table 989.04 Proper rate of centrifuge rotation for known
diameter of wheel to achieve 164 ´ g
Diameter of wheel, cm Diameter of wheel, in. rpm
35.6 14 909
40.6 16 848
45.7 18 800
50.8 20 759
55.9 22 724
61.0 24 693
maintained at 48 ± 1°C, immerse bottle to level slightly above top of
fat column, and leave in H2O bath until column is at equilibrium and
lower fat surface assumes final convex form (≥5 min).
Remove 1 bottle from bath, wipe dry, and with aid of reading light
and magnification, C(k), use dividers or calipers to quickly measure
fat column (before it begins to cool and contract). Place caliper
points in vertical line on neck of bottle, with 1 point at lowest surface
of lower meniscus and other point at top of upper meniscus. Without
changing distance between 2 points on calipers, move calipers down
bottle neck until lower point rests in etched horizontal graduation
mark at 0%. Place upper point of calipers against bottle graduation
and read test in % to nearest 0.05%. Repeat for each bottle.
Fat column, at time of measurement, should be translucent,
golden-yellow or amber, and free of visible suspended particles. Re-
ject all tests in which fat column is milky or shows presence of curd
or charred matter, or in which meniscus is indistinct or distorted; re-
peat test, adjusting volume of H2SO4 added, to obtain proper color
and milk-acid reaction temperature.
References: JAOAC 8, 4(1924); 9, 471(1925); 56, 1401(1973);
58, 949(1975); 71, 898(1988).
J. AOAC Int. 80, 845(1997).
E. Alternative Apparatus
—First Action 1986
—Final Action 1991
(a) Gercock-style milkfat test bottle.—(Available from Forcoven
Products, Inc., PO Box 1556, Humble, TX 77338, USA.) 5%,
162 ± 3 mm high. Bottom of bottle is flat, and axis of flat graduated
tube is vertical when bottle stands on level surface. Quantity of milk
charge is 18 g; 18 g is acid-etched and pigmented in clear space
above 5% graduated line. Specifications:
(1) Bulb.—Capacity of bulb to junction with neck must be
≥45 mL. Shape of bulb is cylindrical, 36 ± 0.5 mm od. Wall thickness
1.4 ± 0.2 mm. Word “Sealed,” manufacturer’s number or name, and
matted marking spot must be legibly and permanently marked on
surface.
(2) Side filling tube.—8 mm od, 0.1 mm wall thickness. To enter
bulb below junction of bulb and neck of flat graduated tube along
curved portion of bulb and with minimum angle extend to center of
bulb and maintain vertical axis for 40 mm to height of 3 ± 2 mm
above bottom of bottle.
(3) Neck.—Graduated portion of flat tube is uniform cross sec-
tion ≥4 mm beyond extremes of graduated scale. Length of gradu-
ated portion of flat neck ≥75 mm. Graduated at whole %, 0.5%,
0.1%, and 0.05%. Graduations are acid-etched, with black pigment
annealed to graduation. Graduation widths ≥0.2 mm. Each whole %
and 0.5% line is numbered on left above line and these lines extend
© 2002 AOAC INTERNATIONAL
across flat surface. Smallest graduated line is ≥2.5 mm. Intermediate Length of nozzle . . . . . . . . . . . . . . . . . . . . 15–20 mm
graduation line extends 1 ± 0.2 mm equally on both sides of smallest Delivery time. . . . . . . . . . . . . . . . . . . . . . . . . 5–8 s
graduated line.
(4) Vertical line.—Vertical line is acid-etched and pigmented on Graduation:
right side of graduated flat tube. This line touches intermediate lines For raw milk. . . . . . . . . . . . . To deliver 17.6 mL at 20°C
at their extreme right end and extends beyond 0 and 0.5% lines when bottom of meniscus coincides
≥3 mm. Width of vertical lines ≤0.2 mm. with mark on suction tube
(5) Error.—Maximum error of total graduation or any part thereof For processsed milk . . . . . . . . . To deliver 17.6 mL at 38°C
must not exceed volume of smallest unit of graduation (= 0.01 mL). when bottom of meniscus coincides
(b) Gercock-style pipet for raw and processed milk.—To con- with mark on suction tube
form to following specifications: Maximum error . . . . . . . . . . . . . . . . . . . . . ≤ 0.05 mL
Permanent markings on bulb:
Total length: For raw milk . . . . . . . . . . . . . . . . . . . . . “Sealed,”
For raw milk . . . . . . . . . . . . . . . . . . . . . . ≤280 mm manufacturer’s No. or name,
For processed milk . . . . . . . . . . . . . . . . . . ≥280 mm TD 17.6 mL at 20°C,
tolerance 0.05 mL, 5–8 s
Suction tube diameter . . . . . . . . . . . . . . . . 7 ± 0.2 mm od
For processed milk . . . . . . . . . . . . . . . . . . “Sealed,”
Wall thickness. . . . . . . . . . . . . . . . . . . . 0.9 ± 0.04 mm manufacturer’s No. or name,
Suction tube length . . . . . . . . . . . . . . . . . . . . 130 mm TD 17.6 mL at 38°C,
Delivery tube diameter . . . . . . . . . . . . . . . 5 ± 0.2 mm od tolerance 0.05 mL, 5–8 s
Distance of graduation mark above bulb . . . . . . . . 15–45 mm
Reference: JAOAC 69, 831(1986).
Revised: March 2002

© 2002 AOAC INTERNATIONAL